1. Field of the Invention
The invention relates to a method for identifying compounds influencing chromosome dynamics in eukaryotic cells. In particular, the invention relates to the treatment and prevention of human conditions by modulating higher order chromatin dependent chromosome stability during mitosis and meiosis.
2. Related Art
Higher-order chromatin is essential for epigenetic gene control and for the functional organisation of chromosomes. Differences in higher-order chromatin structure have been linked with distinct covalent modifications of histone tails which regulate transcriptional xe2x80x98onxe2x80x99 or xe2x80x98offxe2x80x99 states (Grunstein, 1998; Turner, 1998; Strahl and Allis, 2000) and influence chromosome condensation and segregation (Karpen and Allshire, 1997; Wei et al., 1999).
Histones constitute a highly conserved family of proteins (H3, H4, H2A, H2B, H1) which are the major components of eukaryotic chromatin structure. Histones compact genomic DNA into basic repeating structural units, the nucleosomes. In addition to their DNA packaging function, histones have been proven to be integral components of the molecular machinery that regulates gene expression.
Post-translational modifications of histone N-termini, particularly of H4 and H3, are well documented and have functionally been characterised as changes in acetylation (Grunstein, 1998; Turner, 1998; Strahl and Allis, 2000), phosphorylation (Wei et al., 1999) and, most recently, methylation (Chen et al., 1999; Strahl et al., 1999). In contrast to the large number of described histone acetyltransferases (HATs) and histone deacetylases (HDACs), genes encoding enzymatic activities that regulate phosphorylation (Sassone-Corsi et al., 1999; Hsu et al., 2000) or methylation (Chen et al., 1999) of histone N-termini are only beginning to be identified. Moreover, the interdependence of the different histone tail modifications for the integration of transcriptional output or higher-order chromatin organisation is currently not understood.
Overall, there is increasing evidence that the regulation of normal and aberrant cellular proliferation is not only affected on the transcriptional level, but that also a higher level of regulation is involved, i.e. the organisation of chromatin structure through the modification of histone molecules. The determination of the proteins and the molecular mechanisms involved in histone modification will contribute to the understanding of the cellular proliferation program and will thus shed led light on the mechanisms involved in aberrant proliferation occurring in tumour formation and progression (Jacobson and Pillus, 1999).
Genetic screens for suppressors of position effect variegation (PEV) in Drosophila (Reuter and Spierer, 1992) and S. pombe (Allshire et al., 1995) have identified a subfamily of approximately 30-40 loci which are referred to as Su(var)-group (Wallrath, 1998) genes. Interestingly, several histone deacetylases (De Rubertis et al., 1996), protein phosphatase type 1 (Baksa et al., 1993) and S-adenosyl methionine synthetase (Larsson et al., 1996) have been classified as Su(var)s. In contrast, Su(var)2-5 (which is allelic to HP1) (Eissenberg et al., 1992), Su(var)3-7 (Clxc3xa9ard et al., 1997) and Su(var)3-9 (Tschiersch et al., 1994; Schotta and Reuter, 2000) encode heterochromatin-associated proteins. Su(var) gene function thus suggests a model, in which modifications at the nucleosomal level may initiate the formation of defined chromosomal subdomains that are then stabilised and propagated by heterochromatic SU(VAR) proteins (Henikoff, 1997).
Su(var)3-9 is dominant over most PEV modifier mutations (Tschiersch et al., 1994), and mutants in the corresponding S. pombe clr4 gene (Ivanova et al., 1998) disrupt heterochromatin association of other modifying factors and result in chromosome segregation defects (Ekwall et al., 1996). Recently, human (SUV39H1) and murine (Suv39h1 and Suv39h2) Su(var)3-9 homologues have been isolated (Aagaard et al., 1999). It has been shown that they encode heterochromatic proteins which associate with mammalian HP1 (Aagaard et al., 1999). The SU(VAR)3-9 protein family combines two of the most evolutionarily conserved domains of xe2x80x98chromatin regulatorsxe2x80x99: the chromo (Aasland and Stewart, 1995; Koonin et al., 1995) and the SET (Tschiersch et al., 1994; Jenuwein et al., 1998) domain. Whereas the 60 amino acids chromo domain represents an ancient histone-like fold (Ball et al., 1997) that directs eu- or heterochromatic localisations (Platero et al., 1995), the molecular role of the 130 amino acids SET domain has remained enigmatic. Overexpression studies with human SUV39H1 mutants indicated a dominant interference with higher-order chromatin organisation that, surprisingly, suggested a functional relationship between the SET domain and the distribution of phosphorylated (at serine 10) histone H3 (Melcher et al., 2000).
It was an object of the invention to gain further insight into the molecular pathways leading to histone modifications and higher-order chromatin organisation in order to harness these findings for interfering with aberrant gene expression and genomic instability through chromosome mis-segregation and thus provide new cancer therapies.
In particular, it was an object of the invention to investigate the function of members of the SU(VAR)3-9 protein family with the view to develop novel strategies to affect higher-order chromatin dependent chromosome stability. Such strategies can be employed in therapies for the treatment of conditions in which aberrant gene expression and genomic instability through chromosome mis-segregation are causally involved. (The term xe2x80x9cchromosome stabilityxe2x80x9d implies successful segregation of chromosomes resulting in the maintenance of a stable karyotype).
Examples 1 to 7 of the present invention show that mammalian SU(VAR)3-9 related proteins (human SUV39H1, murine Suv39h1 and murine Suv39h2) are SET domain-dependent H3-specific histone methyltransferases which selectively methylate lysine 9 (xe2x80x9cK9xe2x80x9d) of the H3 N-terminus. Methylation of K9 negatively regulates phosphorylation of adjacent serine 10 and reveals a xe2x80x98histone codexe2x80x99 that appears intrinsically linked to the organisation of higher-order chromatin. (In the following, histone methyltransferases are termed xe2x80x9cHMTasesxe2x80x9d or, more generally, xe2x80x9cMTasesxe2x80x9d).
After having identified Suv39h1 and Suv39h2 as mammalian histone H3 lysine 9 specific histone methyltransferases (Suv39h HMTases), it was shown that these HMTases are heterochromatin-enriched enzymes which transiently accumulate at centromeres during mitosis (Aagaard et al., 1999; Aagaard et al., 2000). Moreover, it was shown that methylation of histone H3 at lysine 9 (H3-K9) creates a high-affinity binding site for HP1 proteins (Lachner et al., 2001; Bannister et al., 2001), thereby defining the SUV39H1-HP1 methylation system as a crucial regulatory mechanism for the assembly and propagation of heterochromatin (Jenuwein, 2001). Overexpression of human SUV39H1 induces ectopic heterochromatin and results in chromosome mis-segregation in mammalian cell lines (Melcher et al., 2000). In addition to the essential mitotic functions described above, heterochromatin is also crucial for the dynamic reorganization of meiotic chromosomes. Meiosis is initiated by chromosomal movements from the nuclear lumen to the nuclear envelope, where chromosomes cluster via their pericentric satellite sequences (Hawley et al., 1992; Scherthan et al., 1996). At meiotic prophase, chromosomes condense, followed by homolog pairing and recombination (at pachytene) between maternal and paternal chromosomes. The onset of the meiotic divisions is preceded by desynapsis, further chromosome condensation and histone H3 phosphorylation at pericentric heterochromatin (Cobb et al., 1999). In particular for male germ cells, the haploid genome content is finally organized into one heterochromatic block in elongating spermatids. In Drosophila, heterochromatin and its associated satellite sequences have been proposed to assist in the initial meiotic chromosome movements and in homolog pairing by orienting chromosomes along a similar higher-order structure (Hawley et al., 1992; Karpen et al., 1996; Dernburg et al., 1996b). In germ cells of mammals, a pachytene checkpoint (de Vries et al., 1999) monitors mis-aligned and unpaired chromosomes and arrests cells in meiotic prophase, thereby preventing the production of aneuploid gametes.
It was a further object of the invention to analyse the role of Suv39h1and Suv39h2 in embryonic development and in spermatogenesis in view of utilizing these proteins as drug targets for conditions involving fertility, in particular male fertility.
To solve the problems underlying the present invention, in a first step bioinformatics techniques were applied. Using the SET domains of the SU(VAR)3-9 protein family as a starting alignment, significant sequence and secondary structure similarities (see Methods) to six plant protein methyltransferases were detected.
To investigate whether the SET domain of human SUV39H1 has enzymatic activity, histones were tested as possible substrates for in vitro methylation. The obtained results demonstrate that SUV39H1 harbors an intrinsic histone methyltransferase activity and suggest that this HMTase activity resides in the C-terminal SET domain.
Using recombinant proteins, both murine GST-Suv39h1(82-412) and the corresponding human SUV39H1 fusion protein [GST-SUV39H1(82-412)] were shown to be catalytically active. Short internal deletions were introduced into the two conserved regions of the SET domain core in GST-SUV39H11(82-412), and additional mutants lacking the C-terminal tail (xcex94C-tail) or the SET-associated cysteine-rich region (xcex94cys) were generated. All mutant proteins failed to demonstrate HMTase activity.
Although these results suggest a significant contribution by the cysteine-rich regions, their apparent absence in the plant methyltransferases does not prevent catalytic activity. To investigate enzyme function of the SET domain in more detail, point mutations were introduced into the most highly conserved motif. In vitro HMTase assays indicated that all point mutations, with the exception of one, abolished enzymatic activity. Surprisingly, the latter mutation resulted in an hyperactive enzyme with approximately 20-fold increased activity. The data obtained define the 320 Hxcfx86xcfx86NHSC326 motif in the SET domain as an important catalytic site.
Because the SET domain is one of the most conserved protein motifs in chromatin regulators (Stassen et al., 1995; Jenuwein et al., 1998), it was next analysed whether SU(VAR)3-9 family members or other SET domain proteins contain HMTase activity. GST-fusion products of the extended SET domains of S. pombe CLR4 (Ivanova et al., 1998), human EZH2 (Laible et al., 1997) and human HRX (Tkachuk et al., 1992) were generated that would correspond to GST-SUV39H1(82-412). Interestingly, GST-CLR4(127-490) displayed pronounced HMTase activity at three-to five-fold increased levels as compared to the recombinant SUV39H1 product, consistent with CLR4 carrying an arginine at the hyperactive position. The results obtained from this analysis show, in agreement with the mutational analysis of SUV39H1, that HMTase activity towards free histones appears to require the combination of the SET domain with adjacent cysteine-rich regions, which is a quality found in only a restricted number of SET domain containing proteins.
These experiments indicated that the HMTase activity of mammalian SU(VAR)3-9 related proteins is selective for histone H3 under the chosen assay conditions. To examine this finding in more detail, in vitro methylation reactions were performed with individual histones. It could be shown that H3 is specifically methylated by GST-Suv39h1(82-412), whereas no signals are detected with H2A, H2B or H4. Methylation of H3 has been shown to occur predominantly at lysine 4 in a wide range of organisms, as well as at lysine 9 in HeLa cells, although the responsible HMTase(s) have yet to be defined (Strahl et al., 1999). To investigate the site utilisation profile of Suv39h1, unmodified peptides comprising the wild-type H3 N-terminus and a mutant K9L peptide were tested as substrates. Additionally, insulin and peptides comprising the N-termini of CENP-A (Sullivan et al., 1994), macroH2A (Pehrson and Fried, 1992) were included. These in vitro assays revealed selective methylation of the wild-type H3 peptide. The data obtained also suggested that the H3 N-terminus is a preferred residue for Suv39h1 dependent HMTase activity.
To more definitively determine this site preference, the wild-type H3 N-terminal peptide was in vitro methylated by GST-Suv39h1(82-412), using S-adenosyl-[methyl-3H]-L-methionine. The labelled peptide, purified by reverse-phase HPLC, was then directly microsequenced, and 3H-incorporation associated with each individual amino acid was analysed. The results confirmed selective transfer of methyl-label to lysine 9, demonstrating that Suv39h1 is a highly site-specific HMTase for the H3 N-terminus in vitro.
Murine Suv39h genes are encoded by 2 loci, Suv39h1 and Suv39h2. To investigate the in vivo significance of Suv39h function and Suv39h dependent K9 H3 methylation, mouse strains deficient for both Suv39h1 and Suv39h2 were generated. Suv39h1 and Suv39h2 deficient strains were intercrossed to produce Suv39h double deficient mice. Double mutant mice were born in sub-Mendelian ratios. Some double null embryos exhibited severe growth retardation and exencephaly. In addition surviving double mutants were growth retarded, suggesting a role for Suv39h in cell proliferation.
In order to determine whether the embryonic phenotypes in Suv39h null mice can be attributed to mitotic defects, PMEFs (primary mouse embryonic fibroblasts) derived from Suv39h double mice were analysed. Suv39h double null PMEFs display a reduced G1-index and an increased proportion of cells with aberrant nuclear morphologies, reminiscent of division defects during mitosis. Furthermore, double null cells also show genomic instabilities and readily become aneuploid. The severity of these aneuploidies increases with higher passage numbers. The inability of Suv39h double null cells to maintain a stable karyotype may underlie the Suv39h embryonic phenotype.
Phosphorylation at serine 10 (phosH3) in the N-terminal tail of H3 has been shown to be required for condensation and subsequent segregation of chromosomes (Wei et al., 1999). During the cell cycle, phosH3 initiates within pericentric heterochromatin in late G2 and then progresses along the entire chromosomes during mitosis (Hendzel et al., 1997). It was found that in wild-type PMEFs, approximately 7% of the cells stain positive for the characteristic, heterochromatin-associated phosH3 foci. In contrast, this number is increased by a factor of about 3-fold in Suv39h double null PMEFs. This result suggested that the overall levels of phosH3 may be enhanced in Suv39h double null PMEFs. This was confirmed biochemically. Together, the obtained data are most consistent with a model in which Suv39h-mediated methylation of lysine 9 in H3 negatively regulates phosphorylation of serine 10.
Together, these data clearly demonstrate crucial roles for Suv39h during cell division. Loss of Suv39h function impairs K9 histone H3 metylation and induces defective cell division resulting in genome instabilities. Segregation defects/genome instability underlies the aetiology of many human cancers (Lengauer et al., 1997) and are often a prerequisite for tumour progression. These observations make Suv39h an excellent candidate for novel therapeutic approaches for tumour therapies.
In additon, a set of experiments of the present invention provides in vivo evidence that the absence of Suv39h HMTase activities impairs development and viability of mutant mice, and directly correlates with a nearly complete lack of H3-K9 methylation at pericentric heterochromatin. Notably, Suv39h-deficient mice display chromosomal instabilities in both somatic and meiotic cells that are further evidenced by an increased risk for development of B-cell lymphomas and perturbed chromosome interactions during male meiosis. These in vivo data assign a fundamental role for H3-K9 methylation at pericentric heterochromatin and suggest that the Suv39h HMTases regulate a xe2x80x98heterochromatic competencexe2x80x99 which protects chromosome stability during mitosis and meiosis.
Single gene disruptions for either Suv39h1 or Suv39h2 allow for normal mouse development and do not appear to affect viability and fertility of mutant mice. This apparent redundancy in gene function would be consistent with the overlapping expression profile of the two Suv39h genes during mouse embryogenesis (O""Carroll et al., 2000). By contrast, combined disruption of both genes in Suv39h double null (dn) mice results in severly impaired perinatal viability (≈33%; Table I), growth retardation and hypogonadism in males. Both Suv39h dn males and are infertile. Although Suv39h dn fetuses appear to develop normally until day E12.5, they then display smaller body sizes and frequently are resorbed during late gestation. These in vivo analyses indicate an important role(s) for the Suv39h genes during mammalian development and for overall viability. Since the absence of Suv39h HMTase activities induces genomic instabilities, the high lethality of Suv39h dn fetuses could mainly be a consequence of perturbed chromosome segregation which would significantly impair the proliferation and differentiation programmes of the developing embryo.
Although Suv39h enzymes are the major HMTases for H3-K9 methylation at pericentric heterochromatin in somatic cells (see FIG. 12) and in early meiotic cells (see FIG. 13B), there are xe2x89xa615 unique gene sequences in the mouse genome that contain the evolutionarily highly conserved SET domain and which are likely to encode additional enzymes with putative HMTase activity (Jenuwein, 2001). At least one of these SET domain containing proteins can indeed also methylate H3 at lysine Tachibana et al., 2001 Thus, the ≈33% viability of Suv39h dn mice could be dependent on the compensating activity of other HMTases that may be expressed to varying degrees in the mixed genetic background, in which the Suv39h dn mice have been analyzed.
Absence of Suv39h HMTase activities triggers genomic instabilities in a variety of cell types, including mouse embryonic fibroblasts (PMEFs) (see FIG. 10), fetal liver and bone marrow cells and in spermatogonia (see FIG. 14C). In agreement with the aneuploidies observed in these cellular systems, Suv39h-deficient mice display an increased risk for tumorigenesis, resulting in late-onset B-cell lymphomas in 33% of Suv39h dn mice (see FIG. 11). B-cell lymphomas also develop upon reduced Suv39h gene dosage in compound mutant mice that contain gene disruptions of Suv39h1 (see Table II). Intriguingly, the Suv39h-induced aneuploidies are mainly characterized by segregation failure of the nearly complete set of the chromosomes, resulting in hypo-tetraploid or even hypo-octaploid cells (see FIG. 10). These data suggest a general impairment of chromosome segregation, consistent with the lack of H3-K9 methylation around all acrocentric centromeres in Suv39h dn cells (see FIG. 12).
Distinct modifications of histone N-termini, such as acetylation (Ekwall et al., 1997) and phosphorylation (Wei et al., 1999) have been shown to be required for correct chromosome segregation in S. pombe and Tetrahymena, presumably by inducing a specialised chromatin structure at pericentric heterochromatin that facilitates the establishment of a functional centromere. Because H3-K9 methylation restricts H3 phosphorylation mediated by the Ipl1/aurora kinase (Hsu et al., 2000) and is also interdependent with histone acetylation (Rea et al., 2000), the absence of Suv39h HMTase activities is likely to perturb this distinct histone modification pattern. Second, in addition to altering nucleosome arrangements, histone modifications can generate specific interaction affinities for chromatin-associated proteins (Rice and Allis, 2001). Although the localisation of CENP epitopes appears unaltered, heterochromatic enrichment of HP1 proteins is largely lost in Suv39h dn somatic cells (Lachner et al., 2001). Notably, HP1 interacts in vitro with INCENP (Ainsztein et al., 1998) which forms a complex with aurora-B (Adams et al., 2000; Kaitna et al., 2000). Mutation of INCENP induces severe mitotic abnormalities including macronuclei and internuclear bridges, and results in nearly complete chromosome mis-segregation and cytokinesis failure (Cutts et al., 1999; Adams et al., 2000; Kaitna et al., 2000). These intriguing parallels suggest a possible in vivo link between Suv39h-mediated H3-K9 methylation and aurora-B dependent phosphorylation, and could categorize Suv39h genes as novel tumour suppressor genes.
The Suv39h-mediated chromosomal instabilities only affect a sub-population of cells and do not appear to trigger pronounced apoptosis (see FIG. 10B), consistent with similar analyses of clr4 mutants in S.pombe (Ekwall et al., 1996; Ivanova et at., 1998). These data suggest that the Suv39h-induced defects in somatic cells are not under strict surveillance of known checkpoint controls (Cortez and Elledge, 2000; but see Bernard et al., 1998) and may be caused by rather late segregation problems duting mitosis. Indeed, a fraction of Suv39h dn cells contain chromosomes that lag at anaphase. Since Suv39h dn PMEFs are characterized by hypo-tetraploid and hypo-octaploid karyotypes (see FIG. 10D) and tumor cells contain xe2x80x98butterflyxe2x80x99 chromosomes (see FIG. 11C), a model is proposed (FIGS. 16A,B), in which the absence of H3-K9 methylation would allow stronger or more persistent pericentric associations between aligned metaphase chromosomes. Although a role for the Suv39h HMTases in centromeric cohesion remains to be determined, it provides an attractive mechanism to explain possible cytokinesis failure and mis-segregation of the entire chromosome complement without activating known checkpoint controls.
In contrast to somatic cells, Suv39-mediated defects in male meiosis induce pronounced apoptosis of stage V-VI spermatocytes during the transition from mid to late pachytene (see FIG. 13A). Activation of programmed cell death at this stage has also been observed in mouse mutants that are impaired in DNA damage control (Xu et al., 1996), meiotic recombination (Yoshida et al., 1998; de Vries et al., 1999; Baudat et al., 2000) and synaptonemal complex formation (Yuan et al., 2000). In Suv39h dn spermatocytes, pericentric H3-K9 methylation is specifically reduced at the pre-leptotene stage but, surprisingly, appears as a wild-type staining during later meiotic stages (see FIG. 13B, bottom panel). Thus, in analogy to the increased centromeric associations discussed above, it is proposed that impairment of H3-K9 methylation at the onset of meiosis induces aberrant centromere clustering that can no longer be xe2x80x98rescuedxe2x80x99 by the hypothetical activity of additional H3-K9 HMTases during mid-pachytene. This model (see FIGS. 16A, B) would characterize Suv39h-dependent H3-K9 methylation as one of the earliest requirements to ensure successful meiosis and to prevent illegitimate heterochromatic interactions. Because non-homologous interactions will result in delayed synapsis or even complete pairing failure (see FIG. 14A), they trigger apoptosis by activating the pachytene checkpoint (de Vries et al., 1999), thereby protecting the male germ line from accumulating aneuploidies.
In Suv39h dn mice, spermatogenic failure is promoted by illegitimate chromosomal interactions, synaptic delay, unpaired sex chromosomes and bivalent mis-segregation at meiosis I (see FIGS. 14-15). Notably, a major fraction of these xe2x80x98forbiddenxe2x80x99 interactions comprises physical contacts between the sex chromosomes and autosomes that are largely mediated through centromeric regions (see FIGS. 14D and 14J). These data suggest that the impairment of H3-K9 methylation may allow pericentric heterochromatin to form a more relaxed configuration which is prone to become engaged in random associations. Cytological and genetic studies in Drosophila demonstrated the intrinsic potential of heterochromatin to restrict inter- and intrachromosomal interactions (Dembrug et al., 1996a; Csink and Henikoff, 1996). Moreover, pericentric heterochromatin has been shown to initiate and maintain alignment and pairing of achiasmate chromosomes until meiosis I (Karpen et al.,1996; Dernburg et al.,1996b), suggesting a role for heterochromatin in defining a xe2x80x98self-complementaryxe2x80x99 higher-order chromosome structure that would ensure partner recognition of homologous chromosomes (Karpen et al., 1996). The in vivo data on the function of the Suv39h HMTases would be consistent with these proposed roles of heterochromatin and reveal the first evidence that impaired definition of meiotic heterochromatin can affect chromosome identity in a mammalian organism.
Finally, Suv39h deficiency induces uni-valency of the sex chromosomes at pachytene and at diakinesis (see FIG. 15). Intriguingly, HP1xcex2 (Motzkus et al., 1999; Turner et al., 2000) and the Suv39h2 HMTase (O""Carroll et al., 2000) localise to the specialised chromatin structure of the sex chromosomes in the XY body. Although XY body formation appears normal in early/mid pachytene of Suv39h dn spermatocytes, Suv39h deficiency prolonges H3-K9 methylation (see arrows in FIG. 13B) and induces hypo-condensation of the Y chromosome (see FIG. 15E). These results involve the Suv39h HMTase activities in the definition of the heterochromatic identity of the Y chromosome and suggest that Suv39h-mediated H3-K9 methylation may indirectly promote or stabilise homolog pairing of the heteromorphic sex chromosomes.
Heterochromatin has been first described more than 70 years agao (Heitz, 1928). Because of its stable appearance in the cell nucleus, it has been proposed to serve crucial functions for the inheritance of cell type identities and the fidelity of chromosome segregation. The discoveries of the first HMTases (Rea et al., 2000; O""Carroll et al., 2000) and their mechanistic link to generate a heterochromatic affinity through H3-K9 methylation and recruitment of HP1 proteins (Lachner et al., 2001; Bannister et al., 2001; Nakayama et al., 2001) has now defined an entry point to start dissecting some of the basic roles of heterochromatin.
The experiments of the present invention have provided in vivo evidence that H3-K9 methylation at pericentric heterochromatin is indeed a crucial requirement to ensure mammalian development and to protect chromosome stability in both somatic cells and male germ cells. Because Suv39h deficiency impairs chromosome function in mitosis and meiosis, the data assign a fundamental role for H3-K9 methylation in directing a xe2x80x98heterochromatic competencexe2x80x99 for overall chromosome dynamics and identityxe2x80x94and reveal some of the direct biological functions of the enigmatic entity called heterochromatin.
In a first aspect, the results obtained in the experiments of the present invention show that members of the SU(VAR)3-9 protein family have HMTase activity which identifies them as novel targets for the therapy of proliferative disorders, in particular cancer.
Furthermore, the experiments of the invention demonstrate that the Suv39h HMTases are important for embryonic development and spermatogenesis.
Combined disruption of both Suv39h HMTase genes abolishes H3-K9 methylation at pericentric heterochromatin and induces chromosomal instabilities with an increased risk for tumorigenesis.
In addition, Suv39h double null male mice display complete spermatogenic failure that is largely caused by non-homologous chromosome associations and delayed synapsis, resulting in apoptosis of meiotic prophase cells. Together, these results establish histone H3-K9 methylation as a crucial determinant for pericentric heterochromatin and provide a direct role for the Suv39h HMTases in maintaining a xe2x80x98heterochromatic competencexe2x80x99 that protects chromosome stability during mitosis and meiosis.
The identification of members of the SU(VAR)3-9 protein family, exemplified by human SUV39H1, murine Suv39h1 and murine Suv39h2, as K9 specific histone H3 MTases is the prerequisite for designing assay methods that allow for finding compounds altering, in particular interfering with, higher order chromatin dependent chromosome stability, which is the basis for novel approaches in cancer therapy. (In the following, if not otherwise stated, the term xe2x80x9cSuv39hxe2x80x9d refers to both the murine and the human protein).
Due to the role of Suv39h1 or Suv39h2 in spermatogenesis, compounds modulating the MTase activitiy of these proteins and thus modulating spermatogenesis may also be used in the treatment of male infertility (using compounds that enhance Suv39h MTase activity) and for reversible male contraception (using compounds that inhibit Suv39h MTase activity).
The present invention relates to a method for identifying a compound that alters higher order chromatin dependent chromosome stability during mitosis and meiosis, said method comprising incubating a substrate for a methyltransferase, in the presence of a methyl donor, with a MTase with Suv39h-like MTase activity, in the presence or absence of a test compound and determining whether the compound modulates the MTase activity.
The group of MTases with Suv39h-like MTase activity (in the following also termed xe2x80x9cSuv39h-like MTasesxe2x80x9d) encompasses enzymes which display histone H3 K9 MTase activity or methyltransferase activity for other yet to be identified substrates.
The term xe2x80x9chistone H3 K9xe2x80x9d is not limited to the human SUV39H or mouse Suv39h substrate (i.e. the methylation site of histone H3 at lysine 9), but is meant to encompass any substrate of the histone or histone variant-type of protein, the methylation of which results in the below-defined epigenetic signal.
Additional members of the group of MTases can be identified by bioinformatic/biochemical techniques and tested biochemically. By way of example, in a first step, by searching data bases for similarities, as described in Example 1. Next, an identified candidate can be verified as a MTase with Suv39h-like MTase activity in biochemical assays similar to or identical with those described in the Examples.
This group of Suv39h-like MTases also encompasses MTases with specificities for other histone H3 residues than K9 or for substrates other than histone H3, which are, like the Suv39h K9 histone H3 HMTase activity observed in the present invention, required for higher order chromatin dependent chromosome stability. This epigenetic signal may be a consequence of histone methylation at lysine 9 on H3 alone; however, it cannot be excluded that MTase activity on undefined substrates or a combination of substrate methylation and other covalent modifications, such as phosphorylation or acetylation, at other histone residues are involved.
In the experiments of the present invention, Suv39h variants with point mutations in the SET domain were shown to confer hyperactive HMTase activity to the protein, these Suv39h variants may be advantageously used in the method of the invention.
In a preferred embodiment, the MTase is mouse Suv39h1 or Suv39h2, most preferably, the MTase is human SUV39H1 or SUV39H2.
Since it has been shown in the present invention that recombinant Suv39h retains HMTase activity, most preferably, a recombinant MTase is employed. Suv39h or Suv39h variants can be produced recombinantly according to standard methods by expression in suitable hosts, e.g. bacteria, yeast, insect or eukaryotic cells and purified, e.g. on glutathione-agarose columns if it has been tagged with GST.
The Suv39h1 and SUV39H1 cDNA sequences are known from the literature (Aagaard et al., 1999), the Suv39h2 cDNA sequence is shown in SEQ ID NO:1; the human SUV39H2 cDNA is defined by the ESTs as shown in SEQ ID NO:3-6.
In the case of testing the compounds for their effect on Suv39h activity, the assay comprises, as its essential features, incubating a histone H3 protein or a histone H3 N-terminal fragment including K9, a methyl donor, e.g. methionine or S-adenosyl-L-methionine, with a preparation containing a Suv39h MTase activity and determining MTase activity in the presence or absence of a test substance.
MTase substrates useful in the method of the invention may be those equivalent to or mimicking the naturally occurring substrates, e.g. biochemically purified histone H3, recombinantly produced histone H3, or a histone H3 peptide that contains the K9 methylation site, or other yet to be identified proteins which act as substrates for Suv39h MTases. Novel Suv39h substrates can be identified by bioinformatic and biochemical techniques and tested using the biochemical assays described in the Examples of the present invention. For example, novel Suv39h substrates can be identified by co-immunoprecipitation techniques. Suv39h proteins or tagged versions of Suv39h proteins can be immunoprecipitated with specific antisera and interacting proteins identified by mass spectroscopy techniques. A yeast two hybrid screen using Suv39h proteins or portions of Suv39h proteins as a bait can also be employed to identify novel interacting protein from a variety of cDNA libraries.
In a preferred embodiment, the histone H3 fragment ARTKQTARKSTGGKAPRKQL (SEQ ID NO:7) is employed. Alternatively, a modified peptide may be used for which the MTase has increased affinity/activity. Such peptides can be designed by exchanging and/or adding and/or deleting amino acids and testing the substrate in serial experiments for MTase affinity/activity.
The methyl group of the methyl donor preferably carries a detectable label, e.g. a radioactive or a chromogenic label, which can be quantified upon transfer to the substrate.
Preferably, the methyl donor is radioactively labelled methionine or S-adenosyl-L-methionine.
Alternatively to using a labelled methyl donor, the substrate, upon methylation by the enzyme, is used to serve as an epitope which can be recognised by a specific antibody and hence be quantified by standard immunoassay techniques, e.g. ELISAs. Antibodies useful in this type of assay can be obtained by using the methylated substrate, preferably a small peptide, e.g. the peptide with the sequence shown in SEQ ID NO:7, as an antigen and obtaining polyclonal or monoclonal antibodies according to standard techniques. The generation and purification of a methyl-specific antibody against the histone H3 lysine 9 position is described in the Materials and Methods section. A suitable H3-K9 methyl antibody was also described by Nakayama et al., 2001.
For small scale applications, the screening method can be based on an assay as described in Example 2, 3 or 4.
In an alternative embodiment, the screening method of the invention utilizes the fact that the methylation of histone H3 at lysine 9 (H3-K9) creates a high-affinity binding site for HP1 proteins. In this embodiment, the substrate, upon methylation, is allowed to bind to HP1 and then incubated with a labelled anti-HP1 antibody. The difference in label intensity between the reaction in the absence or presence of the test compound is indicative for the compound""s modulating effect on MTase activity.
HP1 is preferably used in recombinant form. Based on the information of the HP1 cDNA sequence (Jones et al., 2000; Accession No. BC006821), HP1 is produced recombinantly according to standard technology. The recombinant protein or fragments thereof are used to generate polyclonal or monoclonal antibodies that are employed in this assay format.
In a preferred embodiment, the method of the invention is performed on a high-throughput scale. For this embodiment, the major assay components, in particular Suv39h, are employed in recombinant form.
For the high throughput format, the screening methods of the invention to identify MTase inhibitors, are carried out according to standard assay procedures. Such assays are based on the catalytic transfer, mediated by Suv39h or a Suv39h variant, of a methyl group from a donor to a substrate, e.g. a histone H3 peptide. To achieve this, the substrate, e.g. histone H3 or a variant or fragment thereof, is immobilised on a carrier, usually a microtiter plate, and incubated with recombinant Suv39h or a Suv39h variant and a methyl donor.
The methyl group of the methyl donor carries a label, preferably a chromogenic or radioactive label.
Fluorescent or radioactive labels and the other reagents for carrying out the enzymatic reaction on a high-throughput scale are commercially available and can be employed according to the supplier""s instructions (e.g. Molecular Probes, Wallac). Examples for suitable fluorescent labels are coumarin derivatives, e.g., 7-amino-4-methylcoumarin or 7-amino-4-trifluoromethylcoumarin. The radioactive label may be a 14C or a 3H atom. Upon transfer of the methyl group to the substrate by Suv39h, in the case of a chromogenic reagent, the methyl donor changes colour which can be quantified. In the case of using a radioactive methyl donor, the methyl group is transferred to the substrate and can be directly quantified.
The specific assay design depends on various parameters, e.g. on the size of the substrate used. In the the case of using a short peptide, the fluorescence quenching or the fluorescence resonance energy transfer methods are examples for suitable assay technologies, as described below.
The substrate may be tagged, e.g. with biotin, the reaction is then carried out in solution and then transferred to streptavidin coated microtiter plates, e.g. in the case of a radioactive methyl group, xe2x80x9cflashxe2x80x9d plates, the material of which contains the scintillant, or plates which are coated with scintillant. Thus the level of methylation of the substrate can be quantified in a suitable scintillation machine/reader. Alternatively, the assay can be carried out in the streptavidin coated xe2x80x9cflashxe2x80x9d plates with the biotinylated substrate already bound to the plates. This type of assay may also be conducted in the form of a so-called xe2x80x9chomogenous assayxe2x80x9d (an assay type which does not require intermediate transfer and washing steps) e.g. by using microbeads that are coated with scintillant and streptavidin, to which the biotinylated substrate is bound.
Similarly to biotin, other commonly used tags, e.g. Flag, Myc, HA, GST, that are suitable to immobilize the substrate to the plate that is coated with the tag-specific antibody, may be used in the above-described assays.
In a variant, this assay is conducted in the format ELISA type assay; in this case, a methyl-specific antibody is used to detect the amount of methylated substrate bound to the plate.
Alternatively, the plate is coated with an antibody against the methylated substrate to capture the methylated substrate; the substrate is also either tagged or chromogenically labeled and the amount of bound methylated tagged/labeled substrate can be quantified either by a tag-specific antibody or by measuring the level of chromogenic label. By way of example, the substrate is a linear or a branched peptide, e.g. [TARKST]4-K2-K-cys) that is labeled with a chromogenic label, e.g. europium, and upon methylation by a Suv39h-like MTase becomes an epitope for a Lys9-methyl specific antibody (see materials and methods) immobilised on a carrier (e.g. microtiter plate). The non-captured substrate is washed away, the europium label is then cleaved and its fluorescence enhanced and the level of fluorescence is calculated by time resolved fluorescence. The level of fluorescence is directly related to the level of methylated substrate (FIG. 17).
An alternative embodiment is based on the principle that methylation of the peptide may alter its sensitivity to cleavage by a protease. Utilizing this principle, the fluorescence quenching (Resonance Energy Transfer xe2x80x9cRETxe2x80x9d) assay may be employed to determine the amount of methylation of peptidic substrates. In a first step, a Suv39h peptidic substrate, which contains the methylation site and a recognition/cleavage site for a defined protease, that is sensitive to modification (in the particular case, methylation of the lysine) of the recognition/cleavage site, e.g. trypsin or LysC. The peptide carries a fluorescent donor near one end and an acceptor near the other end. In the uncleaved substrate, the fluorescence of the substrate is quenched by the persisting intramolecular RET between donor and acceptor. Upon cleavage of the (unmethylated) substrate by the protease, the cleavage products are released from RET quenching and a fluorescence signal is generated. Methylation of the substrate abolishes the ability of the protease to cleave the substrate. Thus, abolishment of the protease activity (which is proportional to methylation) is reflected by signal repression, in case of total protease inhibtion, total signal repression to the basal level.
An assay of this type may be carried out as follows: the solution of the labeled substrate (e.g. the peptide labeled with 4-[[4xe2x80x2-(dimethylamino)phenyl]azo]benzoic acid (DABCYL) at the one end and with 5-[(2xe2x80x2-aminoethyl)amino]naphtalenesulfonic acid (EDANS) at the other end or labeled with benzyloxycarbonyl at the one end and with 4-aminomethylcoumarin at the other end) in assay buffer is transferred into each well of black 96-well microtiter plates. After addition of the test substances in the defined concentration, the MTase and the methyldonor are added to the wells. After incubation under reaction conditions and for a period of time sufficient for the methylation reaction, e.g. for 40 min at room temperature, the protease, e.g. trypsin, is added and allowed to react under suitable conditions, finally, the fluorescence is measured in a fluorometer at the excitation wavelength, e.g. at 340 nm, and at the emission wavelength, e.g. at 485 nm.
In the case of using the FRET assay, the following commercially availabe labeling pairs are suitable for the method of the invention: Europium (Eu) and Allophycocyanin (APC), Eu and Cy5, Eu and PE (Wallac, Turku, Finland). If a test substance is a modulator of the MTase activity, there will be, depending on the detection system and depending on whether the test substance has an inhibiting or an activating effect, a decrease or an increase in the detectable signal as compared to a control sample in the absence of a test substance. In the high-throughput format, compounds with a modulating effect Suv39h MTase activity can be identified by screening test substances from compound libraries according to known assay principles, e.g. in an automated system on microtiter plates.
By providing a method to identify compounds which exert their effect by directly modulating, in particular by inhibiting, a Suv39h-like MTase, the present invention provides a basis for inhibiting the proliferation of rapidly dividing animal cells, in particular tumour cells.
The compounds identified in the above methods, which are also subject of the invention, have the ability to interfere with chromosome stability and high fidelity chromosome segregation by modulating the MTase activity of Suv39h.
In a preferred embodiment, the compounds of the invention are inhibitors of Suv39h HMTase activity.
Preferably, the compounds are specific modulators of Suv39h, in particular Suv39h1 or Suv39h2.
The present invention also relates to compounds, which act as modulators of a Suv39h-like MTase activity, in particular modulators of Suv39h, for use in human therapy, in particular cancer therapy.
Compounds inhibiting Suv39h HMTase activity result in decreased genome stability and can be used in therapy for targeting dividing cells, in particular highly proliferative tumour cells. They are preferably administered in combination with other genome destabilising agents, e.g. mitose inhibitors like tubulin binders (taxanes, e.g. taxol, Paclitaxel; or epithelones). SUV39H inhibitors may also be used jointly with or before the application of conventional tumour therapies, e.g. radiotherapy or chemotherapy, in particular DNA damaging agents, in order to pre-sensitize the tumour cells. By destabilizing the cell""s genome, the SUV39H inhibitors make the cell more susceptible to the parallel/subsequent treatment.
The SUV39H inhibitors will preferably be used in a combination therapy and applied in consecutive and transient treatments. Since the development of B-cell lymphomas in Suv39h double null mice only occurs with a late onset (i.e. after 9 months of age), transient treatments with SUV39H inhibitors should not induce an immediate increase in tumor risk but rather weaken overall genomic stabilities of highly proliferating cells.
Likewise, agents which enhance Suv39h HMTase activity can be used to stabilise the genome of inherently unstable cells, rendering them less prone to acquiring proliferation promoting mutations. A model for Suv39h function and effects of inhibition or enhancement of Suv39h enzymes is shown in FIG. 8.
The efficacy of compounds identified as Suv39h modulators can be tested for in vivo efficacy in mammalian cells with Suv39h double null cells serving as a positive control. Compounds effective in cancer therapy should interfere with chromosome stability and segregation, which can be measured by karyotyping, e.g. by analysing the DNA content by FACS or standard cytological techniques. Substances whose potential for therapeutic use has been confirmed in such secondary screens can be further tested for their effect on tumour cells. To test the inhibition of tumour cell proliferation, primary human tumour cells are incubated with the compound identified in the screen and the inhibition of tumour cell proliferation is tested by conventional methods, e.g. bromo-desoxy-uridine or 3H thymidine incorporation. Compounds that exhibit an anti-proliferative effect in these assays may be further tested in tumour animal models and used for the therapy of tumours.
Compounds intended for male fertility applications can be tested in animal models described by Vigil et al., 1985, in animal models developed for experimental studies of human spermatogenesis, as described by Weinbauer et al., 2001, or in animal models that mimic human male reproductive defects, as described by Lamb and Niederberger (1994). Guidance for a valid application of animal data to the assessment of human reproductive disorders is given by Working, 1988.
Toxicity and therapeutic efficacy of the compounds identified as drug candidates by the method of the invention can be determined by standard pharmaceutical procedures, which include conducting cell culture and animal experiments to determine the IC50, LD50, the ED50. The data obtained are used for determining the human dose range, which will also depend on the dosage form (tablets, capsules, aerosol sprays, ampules, etc.) and the administration route (oral, buccal, nasal, paterental, rectal or, in the case of temporary male contraceptive applications, local sustained release form applications, e. g. slow-releasing micropellets that are implanted into or adjacent to the gonads). A pharmaceutical composition containing the compound as the active ingredient can be formulated in conventional manner using one or more physologically active carriers and excipients. Methods for making such formulations can be found in manuals, e.g. xe2x80x9cRemington Pharmaceutical Sciencesxe2x80x9d.
As Suv39h is required to maintain a stable karyotype, it can be considered as a tumour suppressor gene. If SUV39H mutations also prove to be a factor underlying cellular transformation events in humans, which is strongly indicated by the analysis of Suv39h double null mice in developing B-cell lymphomas, it can be expected that the re-introduction of a wild type Suv39h gene by gene therapy results in increased genomic stability delaying or inhibiting cancer progression.
For gene therapy, the Suv39h DNA molecules may be administered, preferably contained on a plasmid in recombinant form, directly or as part of a recombinant virus or bacterium. In principle, any method of gene therapy may be used for applying Suv39h recombinant DNA, both in vivo and ex vivo.
Examples of in vivo administration are the direct injection of xe2x80x9cnakedxe2x80x9d DNA, either by intramuscular route or using gene guns. Examples of recombinant organisms are vaccinia virus or adenovirus. Moreover, synthetic carriers for nucleic acids such as cationic lipids, microspheres, micropellets or liposomes may be used for in vivo administration of nucleic acid molecules coding for the Suv39h polypeptide.
Since it has been shown in the present invention that Suv39h mediates dynamic transitions in higher-order mammalian chromatin largely through its intrinsic HMTase activity, histone H3-K9 methylation (H3-K9 Me) represents an important epigenetic imprint for chromosome dynamics during cell division. Hence, antibodies specific for H3-K9 Me can be used to screen cells/patients for heterochromatin based genome instabilities. In essence, H3-K9 methylation specific antibodies can be used as a diagnostic tool for human diseases associated with aberrant gene expression and genomic instability through chromosome mis-segregation or with aberrant definition or organisation of heterochromatin.